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. 2017 Apr 10;7:766. doi: 10.1038/s41598-017-00774-9

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Bulk isolation of optic vesicle-like structures and early characterization in IMR90.4 iPSCs. (a) The treatment leading to optic vesicles and retina cups. (b) Quantitative PCR analysis of OCT4. (c) Cup shaped vesicles at D15 are separated from non-cup shaped vesicles. (d) Viewed at higher magnification, is a 3D retinal cup. (e) At D37 RC’s maintain their cup-like morphology. (f–h) Tissue sections from vesicles probed with antibodies against OTX2 at D35 label retinal progenitors and possibly PRs. (i–k) BRN3+ cells mark RGC’s in the inner retina. (l–n) NF200 labels the entire retina with enhanced labeling of RGC axon bundles. (f,i,l) Hoechst was used as a nuclear counterstain. (o,p,q) Quantitative PCR analysis of the early retinal markers SIX6, OTX2, and BRN3 was performed on cDNA samples pooled (n = 10) from differentiated IMR90.4 iPSCs to confirm their expression. Data from pooled samples (n = 10 per time point) was calculated using three replicates per time point and error bars represent s.e.m. The difference between the indicated time points is statistically significant by Student’s t-test where ***P < 0.001, **P < 0.01, ns, not significant. PR = photoreceptor; RC = retina cup; RGC = retinal ganglion cell. Scale bars (a,b) = 500 μm; (c) = 200 μm; (d) = 100 μm.